Emerging evidence suggest that not all cells of the human body have identical DNA sequence, a phenomenon called somatic mosaicism. Dividing cells can accumulate single nucleotide variations (SNVs) as well as larger structural variants (SVs), such as copy number variations (CNVs). Our recent studies suggest that somatic mosaicism normally occurs in at least 30% of human skin fibroblasts. The human cerebral cortex displays a very high degree of mitotic expansion during ontogenesis and may be particularly susceptible to accumulating somatic variation during development. Somatic mosaicism could be an adaptive or maladaptive phenomenon, accounting for inter-individual human genetic variability and shaping individual susceptibility and resilience to neuropsychiatric disorders. Yet, the extent of somatic mosaicism in the normal human brain is unknown. In this proposal we will investigate the degree of somatic variation in the developing human brain, using postmortem fetal human tissue. The ideal way to study somatic mosaicism would be to sequence the genome of single cells, however, the extreme degree of amplification that is required creates inevitable artifacts. Our principal approach will be to sequence the genome of clonal cell populations derived from single brain cells, identify genomic variants manifested in each clone, and verify the presence and frequency of these variants in the original brain tissue to verify that it is, indeed, mosaic. Using this comprehensive dataset, we will then evaluate and refine variant calls obtained by whole genome amplification of single brain cells. In Aim 1, we will construct a map of somatic variations in human brain progenitor cells and estimate their frequency in the developing cerebral cortex and basal ganglia. We will compare the genomes of clonal cell populations and single cells extracted from brain tissue, followed by high resolution analyses to verify their presence and allele frequency in the original brain tissue as well as in the blood. In Aim 2, we will determine the impact of somatic mosaicism on gene expression by assessing whether clone-manifested genomic variants have consequences at the level of gene transcription and/or have effects on biological functions that may confer adaptive advantage to the cells. In Aim 3, we will investigate the most likely biological origin of somatic variants by analyzing sequence features at variation sites, correlating variants with recombination hotspots, CpG islands and histone marks. Together, these specific aims will provide the first comprehensive estimate of the number and allelic frequency of genomic variation in somatic cells of the brain and will yield hypotheses about mechanisms responsible for their creation as well as their significance for brain development.